Analyses of distal gene expression and proteome in developing tree peony seeds

Tree peony (Paeonia section Moutan DC.) seeds are an excellent source of effective constituents and oil content. In recent years, the tree peony has gained in popularity as a resource in food and nutraceutical industries. Therefore, understanding the transcriptional and translational regulation of seed formation is essential for increasing seed yields and modifying functional components by genetic engineering.In this study, we conducted analysis of distal gene expression and proteome at nine timepoints during the development of tree peony seeds, using a BGISEQ-500 RNA-seq platform and IBT technology. A total of 41808 unigenes were identified from 21.6 billion single-end 50 bp-long reads. Furthermore, 7026 peptides and 2841 proteins were screened out with the 1% false discovery rate (FDR). Our seed samples were divided into four developmental stages according to the cluster analysis of both differentially expressed genes (DEGs) and differentially expressed proteins (DEPs): 25–39 days after pollination (DAP; May 3, May 10 and May 17), 39–67 DAP (May 31 and Jun 14), 67–95 DAP (Jun 28, July 5 and July 12), and 95–109 DAP (July 26). The KEGG Orthology enrichment of DEGs and DEPs demonstrated that May 31 is the critical period for fatty acid biosynthesis and fatty acid metabolism, and alpha-linolenic acid (ALA) shows significant enrichment on July 26. We identified, via KEGG analysis, 211 genes and 35 proteins involved in fatty acid metabolism. By multiple reaction monitoring technology (MRM), the temporal expression patterns of DEPs related to fatty acid metabolism with high expression levels at the active stage of fatty acid metabolism rather than the initial stage of seed development were confirmed.This study provides the first conjoint distal gene expression and proteome analyses of principal molecular events and fatty acid metabolism during developing tree peony seeds. This data not only provide insight into the developmental mechanisms of tree peony seeds, but also reveal novel candidate genes which are worthy of further investigation into the value of genetic engineering of tree peony seeds.牡丹（Paeonia section Moutan DC.，芍药属牡丹组）种子是优质功效成分与油脂的优良来源。近年来，牡丹作为食品及营养保健品行业的原料愈发受到市场青睐。因此，解析种子形成过程中的转录与翻译调控机制，对于提升牡丹种子产量、通过基因工程手段改良其功能成分至关重要。 本研究依托BGISEQ-500 RNA测序（RNA-seq）平台与IBT技术，对牡丹种子发育过程中的9个时间点开展了转录组与蛋白质组分析。从216亿条单端50bp测序读段中，共鉴定得到41808个Unigene（单基因簇）。此外，以1%的错误发现率（False Discovery Rate，FDR）为筛选阈值，共获得7026条肽段与2841个蛋白质。 本研究基于差异表达基因（Differentially Expressed Genes，DEGs）与差异表达蛋白质（Differentially Expressed Proteins，DEPs）的聚类分析结果，将牡丹种子样本划分为四个发育阶段：授粉后25~39天（Days After Pollination，DAP；5月3日、5月10日与5月17日）、授粉后39~67天（DAP；5月31日与6月14日）、授粉后67~95天（DAP；6月28日、7月5日与7月12日）以及授粉后95~109天（DAP；7月26日）。 对DEGs与DEPs进行KEGG直系同源（KEGG Orthology，KO）富集分析结果显示，5月31日是脂肪酸生物合成与脂肪酸代谢的关键时期，而α-亚麻酸（Alpha-Linolenic Acid，ALA）在7月26日呈现显著富集。本研究通过KEGG分析，共鉴定得到211个参与脂肪酸代谢的基因与35个相关蛋白质。 本研究借助多反应监测技术（Multiple Reaction Monitoring，MRM），验证了脂肪酸代谢相关DEPs的时序表达模式：这些蛋白质在脂肪酸代谢活跃期呈现高表达，而非种子发育的初始阶段。 本研究首次针对牡丹种子发育过程中的核心分子事件与脂肪酸代谢，开展了转录组与蛋白质组联合分析。本数据集不仅为解析牡丹种子的发育机制提供了重要理论参考，同时还挖掘出一批潜在候选基因，为后续开展牡丹种子的基因工程改良研究提供了极具价值的研究方向。